Salivary Testosterone

[Michael Scally MD]

Hayes LD, Sculthorpe N, Herbert P, et al. Salivary testosterone measurement does not identify biochemical hypogonadism in aging men: a ROC analysis. Endocrine. http://link.springer.com/article/10.1007/s12020-014-0516-3

The age-associated gradual decline of systemic testosterone (T) production in men is widely known and further intensified by a concomitant increase in concentrations of sex hormone binding globulin (SHBG) with a resultant-pronounced decline in ‘free testosterone’ (free-T) fraction.

The estimated prevalence of clinically low T (biochemical hypogonadism) is not known with certainty for reasons partly due to clinically defined thresholds and variability in measurement. However, a recent report suggested the prevalence to be 0.1 % in men aged 40–49 and 5.1 % aged 70–79 in the European Male Aging Study (EMAS) with other authors suggesting prevalence as high as 49 % in men over 80 years of age.

When considered with report of a possible population-level decline in T, independent of aging, and a World Health Organization (WHO) estimation of 2 billion of the world’s inhabitants being over 60 years by 2050, convenient methods for determining T levels become increasingly attractive.

In recent years, determination of T in saliva has gained popularity as a research tool in the fields of experimental research. This method to determine systemic steroid hormones has advantages over serum including; speed, ease of collection, avoidance of stress, and specialist training associated with venepuncture.

Since the initial determination of T in saliva by Landman and colleagues, studies have reported varying levels of agreement with serum values and in some instances, no relationship at all. It has previously been suggested that salivary testosterone (sal-T) could be used to screen for hypogonadism.

Despite the possible advantages over serum detection of T, numerous analytical and technical issues remain to be resolved. Indeed, sal-T is purported to represent the ‘‘free’’ fraction of T as SHBG is supposedly unable to enter saliva; however, SHBG has been observed in saliva and given that SHBG is known to increase with age, the influence of advancing age on the serum-saliva T relationship is unclear.

In practice, total T (TT) measurement is suggested as the initial test to evaluate male hypogonadism, and therefore, the aim of this study was to determine the discriminative ability of sal-T to predict biochemical hypogonadism based on serum TT in a cohort of aging men.

In conclusion, sal-T demonstrated poor levels of agreement with serum T in older males and ROC analysis revealed that in order to achieve acceptable specificity, sensitivity becomes unacceptably low (22 %).

These data have important implications for the interpretation of sal-T in experimental research and these authors endorse exercising of considerable caution when using sal-T to reflect systemic T in aging men.

 

[Michael Scally MD]

Peres JC, Rouquette JL, Miocevic O, Warner MC, Slowey PD, et al. New Techniques for Augmenting Saliva Collection: Bacon Rules and Lozenge Drools. Clin Ther. http://www.clinicaltherapeutics.com/article/S0149-2918(15)00085-5/abstract

PURPOSE: Saliva is a reliable, noninvasive, and cost-effective alternative to biomarkers measured in other biological fluids. Within certain populations, saliva sampling may be difficult because of insufficient saliva flow, which may compromise disease diagnosis or research integrity. Methods to improve flow rates (eg, administering citric acid, chewing gum, or collecting cotton) may compromise biomarker integrity, especially if the methods involve the presence of a collection aid in the oral cavity. Anecdotal strategies (eg, looking at pictures of food or imagining food) have not been evaluated to date. In this study, we evaluate whether 2 novel collection techniques improve saliva flow or interfere with assay of common biomarkers (ie, cortisol, dehydroepiandrosterone, and testosterone). We evaluate an over-the-counter anhydrous crystalline maltose lozenge intended to increase saliva production for patients with xerostomia long after the lozenge dissolves. We then evaluate whether the smell of freshly cooked bacon stimulates a pavlovian-type reflex.

METHODS: Saliva was collected from 27 healthy young adults (aged 20-34 years; 12 men) on a basal day and a lozenge day, providing 5 samples at 15-minute intervals. Twenty participants then returned for the bacon day condition, providing 2 saliva samples with an interval of 15 minutes between samples. Collection times required to generate 2 mL of saliva across collection strategies were recorded, and then saliva samples were assayed for cortisol, dehydroepiandrosterone, and testosterone.

FINDINGS: Repeated analysis of variance measures revealed that both the lozenges and bacon significantly decreased collection time compared with the passive drool collection on the basal day. No significant effects were found related to the quantification of cortisol, testosterone, or dehydroepiandrosterone when comparing lozenge or bacon to the basal day. In addition, bivariate correlations revealed that concentrations from time-matched control samples correlated significantly with concentrations from the lozenge and bacon conditions.

IMPLICATIONS: These results indicate that both the lozenge and smelling bacon improve saliva collection times and that neither technique interferes with salivary hormone concentrations. This study reveals new methods to augment saliva collection strategies.

 

[MR10X]

"The age-associated gradual decline of systemic testosterone (T) production in men is widely known and further intensified by a concomitant increase in concentrations of sex hormone binding globulin (SHBG) with a resultant-pronounced decline in ‘free testosterone’ (free-T) fraction."

Glad you posted this,my last T levels at 68 are listed below.I wounder if your body senses FT because my LH was so high even though my TT was mid range....My endo even told me your FT to TT ratio changes as you get older.
TT 493 (348-1197)
FT 4.7 (6.6-18.1)
LH 16.1 (1.7-8.6)

 

[Michael Scally MD]

Fiers T, Kaufman JM. Management of hypogonadism: is there a role for salivary testosterone. Endocrine. http://link.springer.com/article/10.1007/s12020-015-0650-6/fulltext.html

In this issue of Endocrine, Lawrence Hayes and colleagues [1] evaluated immunoassays to look at the suitability of salivary testosterone to identify biochemical hypogonadism in aging men. The diagnosis of androgen deficiency in aging men depends on the combination of consistent symptoms and signs and unequivocally low serum testosterone levels determined in a morning blood sample by an accurate assay [2]. Presently, well-validated mass spectrometry-based methods such as isotope dilution gas chromatography–mass spectrometry (ID-GCMS) or liquid chromatography–tandem mass spectrometry (LC-MSMS) are considered the state of the art for testosterone measurement [3]. Since the landmark paper of Taieb first exposed the appalling quality of testosterone immunoassays [4], not much has changed, and numerous papers have since illustrated problematic immunoassay accuracy (IA) and standardization, especially for low values. Data from the EMAS study illustrate that in the context of general screening purposes, IA accuracy can be considered acceptable but accuracy deteriorates in the hypogonadal range [5]. Considering most certified laboratories utilize automated immunoassays, this is sobering knowledge as to the correct analysis and interpretation of a low serum total testosterone result.

For some patients suspected of hypogonadism, the Endocrine Society advocates the use of free or bioavailable testosterone, determined using an accurate method [2]. Most testosterone in the human blood is bound to SHBG and—more weakly—to albumin, and only a few percent is free [6]. With age, in patients with obesity or hyperthyroidism or during corticoid therapy, SHBG levels are markedly influenced. Free testosterone is believed by many authors to be a more informative reflection of testosterone available for biological action, in particular in such situations characterized by altered SHBG levels. Not surprisingly, numerous studies have illustrated stronger associations of clinical parameters with free rather than total testosterone. Free testosterone can be measured using either ultrafiltration or equilibrium dialysis, coupled with a direct or indirect (after the addition of isotope-labeled testosterone) measurement of testosterone in the ultra filtrate or dialysate. The gold standard for free testosterone measurement is direct equilibrium dialysis coupled to mass spectrometry. It remains however out of reach for most laboratories. Equilibrium dialysis is difficult to perform, and direct analysis of the dialysate assumes a highly sensitive and thus high-end LC-MSMS instrument with a thoroughly validated method.

Over the years, alternative solutions have been explored such as direct measurement using immunoassays (inaccurate and should no longer be used) or calculated using total testosterone and SHBG levels [6, 7]. The calculated value has been shown to correlate well with equilibrium dialysis, although some uncertainties remain. A number of authors explored which calculation method works best in different patient cohorts or might mimic more closely the biological binding characteristics of SHBG [7]. Varying bias and scatter versus equilibrium dialysis results have been observed, dependent not only on the calculation methods used, but also on methodological issues related to the equilibrium dialysis (e.g., dilution, standardization, method variability, and indirect estimation using tracer may influence results). The mere fact that calculated free testosterone is a mathematical estimation also undermines its functionality in those patients where normal steroid binding patterns are disturbed (e.g., competition from exogenous steroids, very low or very high SHBG,…).

For some decades now, salivary measurements have been put forward as a non-invasive, stress free, and thus theoretically attractive alternative to steroid measurements in blood. Especially for salivary cortisol extensive literature has been published on its role in population studies, in the follow-up of therapy and, most promising, on a midnight cortisol diagnostic test for Cushing [8]. Immunoassay inherent problems regarding specificity and standardization have been shown to be an even bigger issue for measurements in saliva than serum [9]. Although pre-analytical factors, accuracy, and choice of collection device have all been shown to be important potential confounders, there is now clearly an established place for salivary cortisol measurements.

When looking at testosterone, there is much less compelling evidence. Serum and saliva testosterone concentrations are a 100-fold lower compared to cortisol, and the free percentage in serum is also smaller for testosterone. Therefore, confounding factors such as blood contamination (e.g., oral hygiene) have a much bigger influence on salivary testosterone results compared to that on cortisol [10]. For testosterone, passive drooling is the collection method of choice, whereas for cortisol multiple alternatives offer acceptable results. Also, as male saliva concentrations have been shown to approximate serum free testosterone values, accurate measurement requires state-of-the-art methodology [11, 12].

Up to now, there has been fairly limited clinical experience with salivary testosterone as a biological marker, in contrast to total or free serum testosterone. Possible diurnal variation as compared to serum testosterone has not been well researched. There are no broadly validated reference ranges and the substantial differences in reported values are a stark reminder of those aforementioned (pre-)analytical difficulties in measuring low testosterone concentrations in such a challenging matrix. The risk of both over- and under-diagnosed hypogonadism is therefore much higher.

In this context, the work Lawrence Hayes and colleagues and other investigators performed to explore the accuracy of salivary testosterone to determine androgenic status is timely and important. The study by Hayes is far from conclusive and many questions remain unanswered. It may well be that a place for salivary testosterone in selected cases, such as in population studies or those involving children, can be identified in the future, but only if salivary assays are proven sufficiently sensitive and specific.

Unfortunately given the low concentrations and the above-mentioned specificity issues, it is unlikely that this can be achieved using immunoassays. Well-validated mass spectrometry-based methods are to be recommended. In addition, to these high analytical requirements, there are also challenging biological and pre-analytical potential confounders such as blood contamination, acidification, chewing, absorption,… Thus caution must be expressed in correctly interpreting conflicting reports.

In this context regarding the failure of salivary testosterone to diagnose hypogonadism in the study of Lawrence Hayes and colleagues, it is likely that (pre-)analytical problems have substantially contributed to the negative findings.

Until more evidence in large patient cohorts is gathered using state-of-the-art analytical methods with impeccable pre-analytical care, the current recommendation to rely on a repeated and accurate morning serum total testosterone measurement as a first diagnostic step in men with suspected hypogonadism remains the method of choice in the management of hypogonadism. In those patients with borderline results and suspected alterations in SHBG concentration, assessment of free testosterone can contribute to the diagnosis.

References

1. L.D. Hayes et al., Salivary testosterone measurement does not identify biochemical hypogonadism in aging men: a ROC analysis. Endocrine (2014).

2. S. Bhasin, G.R. Cunningham, F.J. Hayes, A.M. Matsumoto, P.J. Snyder, R.S. Swerdloff, V.M. Montori, Testosterone therapy in adult men with androgen deficiency syndromes: an endocrine society clinical practice guideline. J. Clin. Endocrinol. Metab. 91, 1995–2010 (2006).

3. J.C. Botelho, C. Shacklady, H.C. Cooper, S.S. Tai, K. Van Uytfanghe, L.M. Thienpont, H.W. Vesper, Isotope-dilution liquid chromatography-tandem mass spectrometry candidate reference method for total testosterone in human serum. Clin. Chem. 59, 372–380 (2013).

4. J. Taieb, B. Mathian, F. Millot, M.C. Patricot, E. Mathieu, N. Queyrel, I. Lacroix, C. Somma-Delpero, P. Boudou, Testosterone measured by 10 immunoassays and by isotope-dilution gas chromatography-mass spectrometry in sera from 116 men, women, and children. Clin. Chem. 49, 1381–1395 (2003).

5. I.T. Huhtaniemi et al., Comparison of serum testosterone and estradiol measurements in 3174 European men using platform immunoassay and mass spectrometry; relevance for the diagnostics in aging men. Eur. J. Endocrinol. 166, 983–991 (2012).

6. A. Vermeulen, L. Verdonck, J.M. Kaufman, a critical evaluation of simple methods for the estimation of free testosterone in serum. J. Clin. Endocrinol. Metab. 84, 3666–3672 (1999).

7. M.N. Zakharov, S. Bhasin, T.G. Travison, R. Xue, J. Ulloor, R.S. Vasan, E. Carter, F. Wu, R. Jasuja, A multi-step, dynamic allosteric model of testosterone’s binding to sex hormone binding globulin. Mol. Cell. Endocrinol. 399, 190–200 (2015).

8. H. Raff, Utility of salivary cortisol measurements in Cushing’s syndrome and adrenal insufficiency. J. Clin. Endocrinol. Metab. 94, 3647–3655 (2009).

9. R. Miller, F. Plessow, M. Rauh, M. Gröschl, C. Kirschbaum, Comparison of salivary cortisol as measured by different immunoassays and tandem mass spectrometry. Psychoneuroendocrinology 38, 50–57 (2013).

10. D. Granger, E. Shirtcliff, A. Booth, K. Kivlighan, E. Schwartz, The ‘‘trouble’’ with salivary testosterone. Psychoneuroendocrinology 29, 1229–1240 (2004).

11. T. Fiers, J. Delanghe, G. T’Sjoen, E. Van Caenegem, K. Wierckx, J.M. Kaufman, A critical evaluation of salivary testosterone as a method for the assessment of serum testosterone. Steroids 86, 5–9 (2014).


12. J.S. Mitchell, T.E. Lowe, Matrix effects on an antigen immobilized format for competitive enzyme immunoassay of salivary testosterone. J. Immunol. Methods 349, 61–66 (2009).

 

[Michael Scally MD]

Testosterone in Human Studies: Modest Associations Between Serum and Salivary Measurements

Testosterone is involved in many processes like aggression and mood disorders. As it may easily diffuse from blood into saliva, salivary testosterone is thought to reflect plasma free testosterone level. If so, it would provide a welcome noninvasive and less stressful alternative to blood sampling. Past research did not reveal consensus regarding the strength of the association, but sample sizes were small.

This study aimed to analyse the association in a large cohort. In total, 2,048 participants (age range 18–65 years; 696 males and 1,352 females) were included and saliva (using cotton Salivettes) and plasma were collected for testosterone measurements. Levels were determined by enzyme-linked immunosorbent assay and radioimmunoassay respectively. Free testosterone was calculated by the Vermeulen algorithm. Associations were determined using linear regression analyses.

Plasma total and free testosterone showed a significant association with salivary testosterone in men (adjusted β = .09, p = .01; and β = .15, p < .001, respectively) and in women (adjusted β = .08, p = .004; and crude β = .09, p = .002 respectively). The modest associations indicate that there are many influencing factors of both technical and biological origin.

de Wit AE, Bosker FJ, Giltay EJ, et al. Testosterone in human studies: Modest associations between serum and salivary measurements. Andrologia. 2018;50:e12779. Testosterone in human studies: Modest associations between plasma and salivary measurements